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Trich Laboratory Testing Standardization

Trichomonas foetus DNA testingProper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialObjectivesTo build confidence in Trich testingTo increase consistency in test resultsTo minimize sample quality influence variablesTo minimize laboratory testing variablesTo reduce the burden and cost to man and beastTrich Laboratory Testing StandardizationProper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialOpportunitiesVeterinarian sample collectionVeterinarian sample handling & shipment recommended by LaboratoryLaboratory sample preparation methodLaboratory extract volume usedLaboratory Taq enzyme type usedLaboratory analysis threshold levelLaboratory use of IPC (Internal Positive Control)Laboratory use of USDA licensed kits Laboratory pooling of samplesTrich Laboratory Testing StandardizationProper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialPooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection ofTritrichomonas foetuscolonized bullsLee EffingerLalitha PeddireddiMarilyn SimunichRichard OberstCatherine OConnellIvan Leyva-Baca

Oregon Department of Agriculture, Animal Health and Identification Division, Animal Health Laboratory, Salem, OR (Effinger) Department of Diagnostic Medicine/Pathobiology (Peddireddi), Kansas State University, Manhattan, KS Kansas State Veterinary Diagnostic Laboratory (Oberst), Kansas State University, Manhattan, KS Animal Health Laboratory, Idaho State Department of Agriculture, Boise, ID (Simunich) Animal Health and Food Safety Group at Life Technologies, Austin, TX (Leyva-Baca, OConnell)

JVDI, 2014, Vol. 26(1) 72-87

Proper & ConfidentialThe world leader in serving scienceProprietary & Confidential#Proprietary & ConfidentialProprietary & Confidential4Study Background2010 AAVLD parasitology committee

Proposed a study to determine whether T. foetus samples can be pooled in order to reduce the costs for testing

Lee Effinger from Oregon State Department of Agriculture led Experimental Design for the project

Marilyn Simunich Idaho State Department of Agriculture served as Study Coordinator & Data Keeper

The Life Technologies Animal Health & Food Safety Group agreed to support the studyProper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialStudy ObjectivesDetermine the effect of pooling a single positive sample having various CT ranges with four negative samples (1:5). If a negative effect was seen, a 1:3 pooling study would then be conducted

Compare different sample preparation systems and various real-time PCR (feeder lab workflows) with the 5X MagMAXTM-pathogen RNA/DNA purification kit and amplification with VetMAXTM T. foetus reagents (Life Technologies workflow)

Assess the specificity of the VetMAXTM T. foetus reagents by sequencing all positive samples with CT values less than 38 and suspect sample CT values between 38 and less than 40 cyclesProper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialMaterials and MethodsSample collection (Cultured Smegma Samples)5 Feeder labs provided 803 samples1 on the West Coast1 in the Southwest1 in the Central States 2 in the SouthEach feeder lab ran their own protocol including sample preparation system and real-time PCR

1 Central Study lab (KSVDL)Sample preparation with MagMAXTM Real-time PCR with VetMAXTM T. foetus reagents

Proper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialCultured smegma samples provided by feeder labsSample MatrixSource# of positive samples *# of negative samples*# of inconclusive samples *Total samples submittedCultured smegma samples (A)733010374 (B)28720100 (C)952263 (D)1733050 (F)341820216Total1616402803* As reported by the feeder labsProper & Confidential#Proprietary & ConfidentialProprietary & ConfidentialReal Time PCR Parameters for Feeder LaboratoriesLab ALab BLab C Herds 1-2Lab C Herds 3-6Lab D Lab FStudy LabFinal reaction volume(L)20202525252525Vol. of T. foetus primer/probe per reaction (L)111110.88/1.13-F&R1Volume of extract per reaction(L)4488558Volume Master Mix (L)151512.512.51918.7512.5Primer/probe designMcMillenMcMillenVet-MaxVet-MaxVet-MaxModified McMillenVet-MaxTaq usedUniversal qPCRUniversal qPCRqPCR MMqPCR MMTaqMan Univ.MMAbsolute qPCR low rox mixqPCR MMThermocyclerAB 7500AB 7500Cepheid SCAB7500AB7500AB7500AB7500Thermocycler modeStandardStandardFastStandardStandardStage 1 temperature(C)9595959550/959595Stage 1 time (sec)101060010120/12015600Stage2 denaturation (C)95959597959595Stage 2 denaturation time (sec)1515152201515Stage 2 annealing temp (C)55555555606055Stage 2 annealing time (sec)45454540456045# cycles40404040404540Analysis thresholdFixed 2.0Fixed 2.0Control based threshold-10% max TF/5% max Xeno Control based threshold-10% max TF/5% max XenoFixed 0.2Control based threshold- 10% max TF/XenoAnalysis baseline setting3-153-15autoPositive (Ct)